Pulmonary hypertension (PHT) represents an important clinical problem in the United States. Increased vascular resistance occurs through the combined mechanisms of increased tone and vessel remodeling. Nitric oxide (NO) is an important endogenous vasodilator currently used clinically in ARDS, primary pulmonary hypertension, and persistent pulmonary hypertension of the newborn. Synthesized in the vascular endothelium, its vasorelaxant effects are thought to be primarily mediated through cGMP-dependent reductions in cytosolic [Ca++] in the vascular smooth muscle. A cGMP-dependent kinase (PKG) is thought to be the central figure in cGMP-induced reductions in [Ca++]I. Much of the evidence to support this PKG-dependent pathway has been determined in smooth muscle cells from conduit pulmonary arteries or from other organs, however, and not from the pulmonary circulation that most of the NO/cGMP-dependent relaxation occurs independently of PKG. In addition to activation of PKG, cGMP can also act on membrane bound ion channels. These recently identified ion channels are nonselective to cations and directly bind cGMP and do not require phosphorylation by PKG. The applicants have identified in rat pulmonary microvascular vessels a fragment of this cyclic nucleotide gated (CNG) ion channel using reverse transcriptase/polymerase chain reaction (RT/PCR). The fragment has sequence homology to a cGMP-inhibited ion channel previously identified in the renal epithelium. Given their observation of a cGMP-dependent, but PKG-independent vasorelaxation in the hypertensive pulmonary circulation they hypothesize that this CNG cation channel may be upregulated and activated in PHT. An important component of NO/cGMP dependent vasodilation could involve closure of these channels. This would be consistent with their observations in the hypertensive pulmonary circulation of an increased reliance on NO-dependent, but PKG-independent vasorelaxation. In this proposal they intend to determine the importance and mechanism of this PKG-independent pathway in the pulmonary circulation with particular emphasis on the potential role of the CNG cation channel. Genetic and hypoxia-induced rat models of PHT will be studied. To determine the mechanism of NO/cGMP dependent relaxation they will examine the effect of selective NO, cGMP, and PKG inhibitors on pulmonary vascular resistance in isolated perfused lungs and also on changes in [Ca++]I at the level of the microvascular smooth muscle. To determine the potential role of the CNG channel they will look for evidence of increased gene and protein expression in the hypertensive pulmonary circulation using RT/PCR, ribonuclease protection assay, western blots, and photoaffinity assays. To determine the molecular and biophysical characteristics of the channel they plan to express the full length gene in Chinese hamster ovary cells.